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1.  Increased CD8+ T Cell Response to Epstein-Barr Virus Lytic Antigens in the Active Phase of Multiple Sclerosis 
PLoS Pathogens  2013;9(4):e1003220.
It has long been known that multiple sclerosis (MS) is associated with an increased Epstein-Barr virus (EBV) seroprevalence and high immune reactivity to EBV and that infectious mononucleosis increases MS risk. This evidence led to postulate that EBV infection plays a role in MS etiopathogenesis, although the mechanisms are debated. This study was designed to assess the prevalence and magnitude of CD8+ T-cell responses to EBV latent (EBNA-3A, LMP-2A) and lytic (BZLF-1, BMLF-1) antigens in relapsing-remitting MS patients (n = 113) and healthy donors (HD) (n = 43) and to investigate whether the EBV-specific CD8+ T cell response correlates with disease activity, as defined by clinical evaluation and gadolinium-enhanced magnetic resonance imaging. Using HLA class I pentamers, lytic antigen-specific CD8+ T cell responses were detected in fewer untreated inactive MS patients than in active MS patients and HD while the frequency of CD8+ T cells specific for EBV lytic and latent antigens was higher in active and inactive MS patients, respectively. In contrast, the CD8+ T cell response to cytomegalovirus did not differ between HD and MS patients, irrespective of the disease phase. Marked differences in the prevalence of EBV-specific CD8+ T cell responses were observed in patients treated with interferon-β and natalizumab, two licensed drugs for relapsing-remitting MS. Longitudinal studies revealed expansion of CD8+ T cells specific for EBV lytic antigens during active disease in untreated MS patients but not in relapse-free, natalizumab-treated patients. Analysis of post-mortem MS brain samples showed expression of the EBV lytic protein BZLF-1 and interactions between cytotoxic CD8+ T cells and EBV lytically infected plasma cells in inflammatory white matter lesions and meninges. We therefore propose that inability to control EBV infection during inactive MS could set the stage for intracerebral viral reactivation and disease relapse.
Author Summary
There is general consensus that multiple sclerosis (MS) is associated with Epstein-Barr virus (EBV) infection but the mechanistic links are still debated. EBV is a B-lymphotropic herpesvirus widespread in the human population and normally contained as a persistent, asymptomatic infection by immune surveillance. However, EBV can cause infectious mononucleosis, is associated with numerous human malignancies, and is implicated in some common autoimmune diseases. While EBV infection alone cannot explain MS development, it has been postulated that in susceptible individuals alterations in the mechanisms regulating the immune response to the virus may contribute to MS pathogenesis. Here, we show that MS patients with inactive disease exhibit a lower CD8+ T-cell response to EBV when compared to healthy donors and active MS patients while the latter have a higher frequency of CD8+ T cells specific for EBV lytic antigens. Therapy with interferon-β and natalizumab, two treatments for relapsing-remitting MS, was associated with marked changes in the EBV specific CD8+ T cell response. We also demonstrate that one of the EBV lytic antigens recognized by CD8+ T cells expanding in the blood during active MS is expressed in the inflamed MS brain. Our results support a model of MS pathogenesis in which EBV infection and reactivation in the CNS stimulates an immunopathological response and suggest that antiviral or immunomodulatory therapies aimed at restoring the host-EBV balance could be beneficial to MS patients.
PMCID: PMC3623710  PMID: 23592979
2.  Absence of Epstein-Barr virus in the brain and CSF of patients with multiple sclerosis(e–Pub ahead of print) 
Neurology  2010;74(14):1127-1135.
Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that becomes latent in B-lymphocytes and has been implicated in the pathogenesis of multiple sclerosis (MS). We searched for latent and active EBV infection in MS brain and CSF.
Nested and non-nested real-time PCR were used to detect cell-specific and EBV-specific transcripts in 15 fresh-frozen and 5 formalin-fixed paraffin-embedded MS plaques and in single MS CSF B-lymphocytes and plasma cells. Intrathecal anti-EBV antibody synthesis was measured by ELISA. Immunocytochemistry was used to detect binding of MS CSF and recombinant antibodies (rAbs) generated from clonally expanded plasma cells in MS CSF to EBV-infected cells.
No EBV RNA was found in MS CSF B-lymphocytes or plasma cells. In active MS plaques, EBV-encoded RNA (EBER)-1 was the only and rarely detected transcript. The frequency of detected intrathecal anti-EBV antibody synthesis in patients with MS did not differ from that in non-MS inflammatory CNS disease control patients. Anti-EBV antibodies were detected in the CSF of patients with MS, but MS rAbs did not react with EBV.
Application of real-time PCR to multiple sclerosis brain and single B-lymphocytes in CSF did not reveal any evidence of active Epstein-Barr virus infection.
= antibody index;
= EBV-encoded RNA;
= Epstein-Barr virus;
= non-MS inflammatory CNS disease;
= immunoglobulin G;
= in situ hybridization;
= Luxol fast blue;
= multiple sclerosis;
= odds ratio;
= recombinant antibodies.
PMCID: PMC2865779  PMID: 20220124
3.  Differences in Gastric Carcinoma Microenvironment Stratify According to EBV Infection Intensity: Implications for Possible Immune Adjuvant Therapy 
PLoS Pathogens  2013;9(5):e1003341.
Epstein-Barr virus (EBV) is associated with roughly 10% of gastric carcinomas worldwide (EBVaGC). Although previous investigations provide a strong link between EBV and gastric carcinomas, these studies were performed using selected EBV gene probes. Using a cohort of gastric carcinoma RNA-seq data sets from The Cancer Genome Atlas (TCGA), we performed a quantitative and global assessment of EBV gene expression in gastric carcinomas and assessed EBV associated cellular pathway alterations. EBV transcripts were detected in 17% of samples but these samples varied significantly in EBV coverage depth. In four samples with the highest EBV coverage (hiEBVaGC – high EBV associated gastric carcinoma), transcripts from the BamHI A region comprised the majority of EBV reads. Expression of LMP2, and to a lesser extent, LMP1 were also observed as was evidence of abortive lytic replication. Analysis of cellular gene expression indicated significant immune cell infiltration and a predominant IFNG response in samples expressing high levels of EBV transcripts relative to samples expressing low or no EBV transcripts. Despite the apparent immune cell infiltration, high levels of the cytotoxic T-cell (CTL) and natural killer (NK) cell inhibitor, IDO1, was observed in the hiEBVaGCs samples suggesting an active tolerance inducing pathway in this subgroup. These results were confirmed in a separate cohort of 21 Vietnamese gastric carcinoma samples using qRT-PCR and on tissue samples using in situ hybridization and immunohistochemistry. Lastly, a panel of tumor suppressors and candidate oncogenes were expressed at lower levels in hiEBVaGC versus EBV-low and EBV-negative gastric cancers suggesting the direct regulation of tumor pathways by EBV.
Author Summary
Epstein-Barr virus (EBV) is detected in roughly 10% of gastric carcinoma (GC) cases worldwide. Despite a strong link between EBV and gastric carcinoma, the contribution of EBV to the tumor environment in EBV associated gastric carcinoma is unclear. We performed a global assessment of EBV and host cell gene expression in gastric carcinoma tumors from 71 patients to link EBV genes (and expression intensities) to cell and microenvironmental changes. In addition to the finding that EBV is associated with down-regulated tumor regulatory genes, this study revealed that samples with high levels of EBV gene expression (hiEBVaGCs) displayed elevated immune cell infiltration with high interferon-gamma (IFNG) expression compared to samples with low or no EBV gene expression. Despite this evidence of increased immune posturing, hiEBVaGC samples also showed elevated expression of the potent immune cell inhibitor, IDO1. This finding may partly explain the persistence of these virus associated tumors in the face of local immune cell concentration. Importantly, the small molecule IDO inhibitor, 1MT (1-methyl Tryptophan), has been shown to reverse the tolerance inducing effects of IDO1 in other tumors. We propose that stratification of gastric carcinomas into EBV-negative, EBV-low and EBV-high may provide indicator value for the use of IDO1 inhibitors as adjuvant therapies against hiEBVaGCs.
PMCID: PMC3649992  PMID: 23671415
4.  Humoral immune response to EBV in multiple sclerosis is associated with disease activity on MRI 
Neurology  2009;73(1):32-38.
Evidence suggests that Epstein-Barr virus (EBV) plays a role in triggering or perpetuating disease activity in multiple sclerosis (MS).
We investigated 100 subjects (50 clinically isolated syndrome [CIS], 25 relapsing-remitting [RR] MS, 25 primary progressive [PP] MS) for 1) evidence of EBV reactivation and 2) disease activity as indicated by serial gadolinium (Gd)-enhanced MRIs over a 5-year period. EBV DNA in blood was quantified by real-time quantitative PCR and EBV serology for anti-Epstein-Barr virus nuclear antigen 1 (EBNA-1) immunoglobulin G (IgG), anti-viral capsid antigen (VCA) IgG, and anti-EBV IgM. Data were analyzed using repeated measures analysis, analysis of variance, and logistic regression analysis.
All subjects had serologic evidence of previous EBV infection, but no lytic reactivation was detected. Significant differences in EBNA-1 IgG titers were found between subgroups, highest in the RRMS cohort compared with PPMS (p < 0.001) and CIS (p < 0.001). Gd-enhancing lesions on MRI correlated with EBNA-1 IgG (r = 0.33, p < 0.001) and EBNA-1:VCA IgG ratio (r = 0.36, p < 0.001). EBNA-1 IgG also correlated with change in T2 lesion volume (r = 0.27, p = 0.044) and Expanded Disability Status Scale score (r = 0.3, p = 0.035).
The correlation between elevated Epstein-Barr virus nuclear antigen 1 (EBNA-1) immunoglobulin G (IgG) and gadolinium-enhancing lesions suggests an association between Epstein-Barr virus (EBV) infection and multiple sclerosis (MS) disease activity. The heightened immune response to EBV in MS is specifically related to EBNA-1 IgG, a marker of the latent phase of the virus. The lack of association between acute viral reactivation in the peripheral blood and Gd+ lesions suggests a limited role of the former in driving disease activity.
= confidence interval;
= clinically isolated syndrome;
= cytomegalovirus;
= Epstein-Barr virus nuclear antigen 1;
= Epstein-Barr virus;
= Expanded Disability Status Scale score;
= field of view;
= gadolinium-DTPA;
= human leukocyte antigen;
= immunoglobulin G;
= interleukin;
= interquartile range;
= major histocompatibility complex;
= multiple sclerosis;
= oligoclonal IgG bands;
= odds ratio;
= phocine herpesvirus type 1;
= primary progressive multiple sclerosis;
= relapsing-remitting multiple sclerosis;
= echo time;
= repetition time;
= viral capsid antigen;
= varicella zoster virus.
PMCID: PMC2848585  PMID: 19458321
5.  Characteristics of Epstein-Barr virus-associated gastric cancer: A study of 235 cases at a comprehensive cancer center in U.S.A 
Epstein-Barr virus (EBV) has been shown to be associated with gastric cancer. However, inconsistent findings have been reported regarding the distribution of EBV infected cells (in normal gastric epithelium vs. intestinal metaplastic cells vs. in neoplastic cells) and the characteristics of EBV-associated gastric cancer. Lymph node positive EBV-associated gastric cancer has not been systematically studied. The aims of this study were to evaluate EBV-associated gastric cancer, to assess the distribution of EBV infected cells including all positive lymph nodes, and to define the characteristics of EBV-associated gastric cancer.
The study included primary gastric cancer patients who underwent surgical resection with no preoperative treatment at M.D. Anderson Cancer Center between 1987 and 2006. Formalin-fixed paraffin-embedded tissue from these resection specimens were assessed for EBV by in situ hybridization, the gold standard for EBV detection in tissue. EBV status was analyzed along with clinicopathologic parameters including age, gender, tumor type, lymph node status, and pathologic stage of the tumor.
Among 235 patients, 12 had intranuclear expression of EBV. EBV staining was seen only in tumor cells and no detectable EBV was observed in normal gastric mucosa, intestinal metaplasia or stromal cells. Eight of 12 patients with EBV-associated gastric cancer had regional lymph node metastasis. Of note, metastatic tumor cells in all of the involved lymph nodes of these 8 cases contained EBV. The epidemiologic data showed 11 of the 12 patients with EBV-associated gastric cancer were men, ranging in age from 54 to 78 years (mean age, 60 years; median age, 62.1 years). The age distribution for non-EBV associated gastric cancer patients ranged from 21 to 93 years (mean age, 67 years; median age, 66.4 years).
Our study demonstrated that EBV is present exclusively in gastric cancer cells. The detection of EBV in tumor cells in all of the lymph nodes involved with metastatic gastric carcinoma suggests simultaneous replication of EBV and tumor cells. The predominantly male gender and relatively younger age observed for the EBV-infected gastric cancer cases suggest an association between this disease and other factors, such as life style.
PMCID: PMC2642773  PMID: 19192297
6.  Dysregulated Epstein-Barr virus infection in the multiple sclerosis brain 
The Journal of Experimental Medicine  2007;204(12):2899-2912.
Epstein-Barr virus (EBV), a ubiquitous B-lymphotropic herpesvirus, has been associated with multiple sclerosis (MS), an inflammatory disease of the central nervous system (CNS), but direct proof of its involvement in the disease is still missing. To test the idea that MS might result from perturbed EBV infection in the CNS, we investigated expression of EBV markers in postmortem brain tissue from MS cases with different clinical courses. Contrary to previous studies, we found evidence of EBV infection in a substantial proportion of brain-infiltrating B cells and plasma cells in nearly 100% of the MS cases examined (21 of 22), but not in other inflammatory neurological diseases. Ectopic B cell follicles forming in the cerebral meninges of some cases with secondary progressive MS were identified as major sites of EBV persistence. Expression of viral latent proteins was regularly observed in MS brains, whereas viral reactivation appeared restricted to ectopic B cell follicles and acute lesions. Activation of CD8+ T cells with signs of cytotoxicity toward plasma cells was also noted at sites of major accumulations of EBV-infected cells. Whether homing of EBV-infected B cells to the CNS is a primary event in MS development or the consequence of a still unknown disease-related process, we interpret these findings as evidence that EBV persistence and reactivation in the CNS play an important role in MS immunopathology.
PMCID: PMC2118531  PMID: 17984305
7.  Evidence of Epstein-Barr Virus Association with Gastric Cancer and Non-Atrophic Gastritis 
Viruses  2014;6(1):301-318.
Different lines of evidence support an association between Epstein-Barr virus (EBV) and gastric cancer (GC). The main understood risk factor to develop GC is infection by Helicobacter pylori (H. pylori), which triggers a local inflammatory response critical for progression from gastritis to GC. The role of EBV in early inflammatory gastric lesions has been poorly studied. A recent study proposed a cutoff value of 2000 EBV particles to identify patients with increased chances of infection of the gastric epithelium, which may favor the inflammatory process. To better understand the role of EBV in cancer progression, we analyzed 75 samples of GC, 147 control samples of non-tumor gastric tissue derived from GC patients and 75 biopsies from patients with non-atrophic gastritis (NAG). A first-round PCR was used for EBV detection in tumor and non-tumor controls and a more sensitive nested PCR for gastritis samples; both PCRs had lower detection limits above the proposed cutoff value. With this strategy 10.67% of GC, 1.3% of non-tumor controls and 8% of gastritis samples were found positive. An EBER1 in situ hybridization showed EBV infection of epithelial cells in GC and in a third of NAG samples, while in the other NAGs infection was restricted to the mononuclear cell infiltrate. EBV-positive GCs were enriched in lace and cribriform patterns, while these rare patterns were not observed in EBV negative samples. Our results support a role for EBV in GC and early precursor lesions, either as directly oncogenic infecting epithelial cells or indirectly as an inflammatory trigger.
PMCID: PMC3917444  PMID: 24448220
Epstein-Barr virus; EBV; gastric cancer; non-atrophic gastritis; cribriform pattern and lace pattern
8.  Expression of EBV Encoded viral RNA 1, 2 and anti-inflammatory Cytokine (interleukin-10) in FFPE lymphoma specimens: a preliminary study for diagnostic implication in Pakistan 
Diagnostic Pathology  2011;6:70.
Epstein Barr Virus (EBV) plays a significant role as a cofactor in the process of tumorigenesis and has consistently been associated with a variety of malignancies. EBV encoded RNAs (EBER1 and EBER2) are the most abundant viral transcripts in latently EBV-infected cells and their role in viral infection is still unclear. Formalin Fixed Paraffin Embedded (FFPE) tissues of surgically removed carcinoma biopsies are widely available form but have never been exploited for expressional studies previously in Pakistan. Immunohistochemistry (IHC) and in situ hybridization (ISH) in FFPE biopsy tissues remains the gold standard for proving EBV relationship in a histopathological lesion but their reagents associated limitations confines their reliability in some applications. Recently introduced targeted drug delivery systems induce viral lytic gene expression and therefore require more sensitive method to quantify viral as well as cellular gene expression.
Eight (8) lymphoma samples were screened to detect the EBV genome. Qualitative and quantitative expression of EBV Encoded RNAs (EBER1, EBER2) and anti-inflammatory cytokine (interleukin-10) in FFPE EBV positive lymphoma tissue samples were then analysed by using Reverse transcriptase Polymerase Chain Reaction (RT-PCR) and Real Time Polymerase Chain Reaction (qRT-PCR), respectively.
In this study we have successfully quantified elevated expressional levels of both cellular and viral transcripts, namely EBER1, EBER2 and anti-inflammatory cytokine (IL-10) in the FFPE Burkitt's lymphoma (BL) specimens of Pakistani origin.
These results indicate that FFPE samples may retain viral as well as cellular RNA expression information at detectable level. To our knowledge, this is first study which represents elevated expressional levels of EBER1, EBER2 and IL-10 in FFPE tissue samples of Burkitt's lymphoma in Pakistan. These observations will potentially improve current lacunas in clinical as well as diagnostic practices in Pakistan and can be further exploited to develop new strategies for studying cellular and/or viral gene expression.
PMCID: PMC3157411  PMID: 21791113
9.  A Case of Age-Related Epstein–Barr Virus (EBV)-Associated B Cell Lymphoproliferative Disorder, so-called Polymorphous Subtype, of the Mandible, with a Review of the Literature 
Head and Neck Pathology  2012;7(2):178-187.
Epstein–Barr virus (EBV) is known to be associated with the development of lymphomas in immunocompromised patients. Recently, age-related immune impairment has been recognized as a predisposing factor in the development of EBV-driven lymphoproliferative disorders (LPDs) in elderly patients without any known immunodeficiency or prior lymphoma. In approximately 70 % of reported cases, the affected sites have been extranodal, such as the skin, lung, tonsil and stomach. However, age-related EBV-associated B cell (EBV + B cell) LPD is extremely rare in the oral cavity. Here we report a 71-year-old Japanese man who developed an EBV + B cell LPD resembling classical Hodgkin lymphoma (CHL)—so-called polymorphous subtype—of the mandible. Histopathologically, infiltration of large atypical lymphoid cells including Hodgkin or Reed-Sternberg-like cells into granulation tissue with marked necrosis was found in the mandibular bone. Immunohistochemical analysis revealed that the large atypical Hodgkin or Reed-Sternberg-like cells were CD3–, CD15–, CD20+, CD30+ and Epstein–Barr virus (EBV)-latent infection membrane protein-1 (LMP-1)+. In situ hybridization (ISH) demonstrated EBV-encoded small RNA (EBER) + in numerous Hodgkin or Reed-Sternberg-like cells. EBNA-2 was detected by polymerase chain reaction (PCR) using an extract from the formalin-fixed, paraffin-embedded specimen. To our knowledge, this is the first reported case of the polymorphous subtype of age-related EBV + B cell LPD affecting the mandible.
PMCID: PMC3642255  PMID: 22869357
Age-related Epstein–Barr virus (EBV)-associated B cell lymphoproliferative disorder (age-related EBV + B cell LPD); Epstein–Barr virus (EBV); Polymorphous subtype; Mandible
10.  Expression of HLA Class I and HLA Class II by Tumor Cells in Chinese Classical Hodgkin Lymphoma Patients 
PLoS ONE  2010;5(5):e10865.
In Caucasian populations, the tumor cells of Epstein Barr virus (EBV)-positive classical Hodgkin Lymphomas (cHL) patients more frequently express HLA class I and HLA class II molecules compared to EBV-negative cHL patients. HLA expression (in relation to EBV) in Asian cHL patients has not been previously investigated.
Methodology/Principal Findings
We randomly selected 145 cHL patients with formalin-fixed, paraffin embedded tissue blocks available from 5 hospitals from the Northern part of China. Hematoxylin & Eosin-stained slides were used to reclassify the histological subtypes according to the WHO classification. EBV status was determined by visualization of EBERs in tumor cells using in situ hybridization. Membranous expression of HLA molecules was detected by immunohistochemistry using antibodies HC-10 (class I heavy chain) and anti-ß2-microglobulin for HLA class I, and CR3/43 for HLA class II. EBV+ tumor cells were observed in 40% (58/145) of the cHL patients. As expected, the percentage of EBV+ cases was much higher in the mixed cellularity subtype (71%) than in the nodular sclerosis subtype (16%) (p<0.001). Expression of HLA class I was observed in 79% of the EBV+ cHL cases and in 30% of the EBV- cases (p<0.001). For HLA class II, 52% of EBV+ cHL cases were positive, compared to 43% in EBV- cases (p = 0.28).
The results in the Northern China population were similar to those in the Caucasian population for HLA class I, but not for HLA class II.
PMCID: PMC2878318  PMID: 20526359
11.  Gammaherpesvirus Latency Accentuates EAE Pathogenesis: Relevance to Epstein-Barr Virus and Multiple Sclerosis 
PLoS Pathogens  2012;8(5):e1002715.
Epstein-Barr virus (EBV) has been identified as a putative environmental trigger of multiple sclerosis (MS), yet EBV's role in MS remains elusive. We utilized murine gamma herpesvirus 68 (γHV-68), the murine homolog to EBV, to examine how infection by a virus like EBV could enhance CNS autoimmunity. Mice latently infected with γHV-68 developed more severe EAE including heightened paralysis and mortality. Similar to MS, γHV-68EAE mice developed lesions composed of CD4 and CD8 T cells, macrophages and loss of myelin in the brain and spinal cord. Further, T cells from the CNS of γHV-68 EAE mice were primarily Th1, producing heightened levels of IFN-γ and T-bet accompanied by IL-17 suppression, whereas a Th17 response was observed in uninfected EAE mice. Clearly, γHV-68 latency polarizes the adaptive immune response, directs a heightened CNS pathology following EAE induction reminiscent of human MS and portrays a novel mechanism by which EBV likely influences MS and other autoimmune diseases.
Author Summary
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that leads to progressive disability. The causes of the disease are still unknown. Viral infections has been linked to MS development and Epstein-Barr virus has been shown to have a strong link to MS. Here, we use an animal model of MS, experimental autoimmune encephalitis (EAE), to study how EBV can trigger MS. Since EBV does not infect rodents, we infected mice with murine gamma herpesvirus 68 (γHV-68), the murine equivalent of EBV. We found that mice previously infected with γHV-68 developed more severe EAE when compared to uninfected EAE mice and showed pathological features that recapitulate human MS. γHV-68 EAE presented with increased CD4 and CD8 T cells infiltrations in the brain, increased brain inflammation and demyelination. This model provides new insight on how EBV might be triggering MS, shedding light on the development of the disease.
PMCID: PMC3355105  PMID: 22615572
12.  Study of Epstein-Barr virus expression in Burkitt's lymphoma by polymerase chain reaction and in situ hybridization: A study in Iran 
Dental Research Journal  2014;11(3):380-385.
The association of Epstein-Barr virus (EBV) with Burkitt's lymphoma (BL) is variable in different geographic regions. In developing countries, the association of EBV with BL is regarded to be of an endemic-type in equatorial Africa (> 95%) and sporadic-type in the developed countries (15-30%). The purpose of this study is to assess the frequency of EBV infection in BL, in Iran. The study also aims to compare Ribonucleic acid (RNA) in situ hybridization (RISH), the standard diagnostic method, with the polymerase chain reaction (PCR)-based method for diagnosing BL.
Materials and Methods:
In this epidemiological study, the paraffinized specimens of 18 cases of BL were selected. Next, the ISH of EBV-encoded RNA (EBER-RISH) and PCR assays that were based on Epstein Barr Nuclear Antigen 2 (EBNA2) amplification were used. The EBV strain was determined by PCR. The data were analyzed using the SPSS10 software and by performing Pearson correlation coefficient formula at a significant level of 0.05.
EBV RNA was detected in 50% of the BL specimens. Type 1 and 2 accounted for 70 and 30% of the cases, respectively. Regarding RISH as the standard method for EBV diagnosis, the PCR assays showed a sensitivity and specificity of 100 and 88.9%, respectively.
According to the obtained findings, the frequency of EBV in BL was 50% and PCR and RISH showed high concordance and sensitivity in EBV detection. Therefore, PCR can be used as a faster method for EBV detection in high-risk geographical regions.
PMCID: PMC4119373  PMID: 25097650
Burkitt's lymphoma; Epstein-Barr virus; in situ hybridization; polymerse chain reaction
13.  Correlation of Epstein-Barr virus and its encoded proteins with Helicobacter pylori and expression of c-met and c-myc in gastric carcinoma 
AIM: To investigate the interrelationship of Epstein-Barr virus (EBV) and EBV- encoded proteins with Helicobacter pylori (H pylori) infection and the expression of c-met and c-myc oncogene proteins in gastric carcinoma, and to explore their role in gastric carcinogenesis.
METHODS: One hundred and eighty-five gastric carcinoma tissues were detected by polymerase chain reaction (PCR)-Southern blot for EBV genome and in situ hybridization (ISH) for EBV-encoded small RNA 1 (EBER1). Gastric carcinoma with positive EBER1 signals was confirmed EBV-associated gastric carcinoma (EBVaGC). The status of H pylori infection in 185 gastric carcinomas was assessed by rapid urease test and PCR. The samples with positive PCR and urease test were defined as H pylori infection. The expression of c-met and c-myc oncogene proteins in tissues of EBVaGC and matched EBV-negative gastric carcinoma (EBVnGC) were examined by immunohistochemistry. RT-PCR and Southern hybridization were used to detect the expression of nuclear antigens (EBNAs) 1 and 2, latent membrane protein (LMP) 1, early genes BARF1 and BHRF1 in EBVaGC cases.
RESULTS: The positive rate of H pylori and EBV in 185 gastric carcinomas was 59.45% (110/185) and 7.03% (13/185) respectively. No difference was found in sex, age, pathological differentiation, clinical stages and lymph node metastasis between H pylori-positive and H pylori-negative gastric carcinomas. However, the positive rate of H pylori infection in the antrum gastric carcinomas was higher than that of cardia and body gastric carcinomas. In our series, age, pathological differentiation, clinical stages, lymph node metastasis and location of cancer were not different between EBVnGC and EBVaGC, while the positive rate of EBV in male patients was significantly higher than that of female patients. The positivity of H pylori in EBV-associated and EBV-negative gastric carcinomas was 46.15% (6/13) and 81.40%(104/172) respectively. There was no significant correlation between EBV and H pylori infection. The c-met overexpression was significantly higher in the EBVaGC group than in the EBVnGC group. However, c-met and c-myc expression did not show significant difference between the two groups. Transcripts of EBNA1 were detected in all 13 EBVaGCs, while both EBNA2 and LMP1 mRNA were not detected. Six of the 13 cases exhibited BARF1 transcripts and 2 exhibited BHRF1 transcripts.
CONCLUSION: The positivity of H pylori in EBVnGCs is higher than that of EBVaGCs, but no significant correlation is found between EBV infection and H pylori infection. H pylori-positive gastric carcinoma is predominant in antrum location, while EBVaGC has a tendency of predominance in cardia/body location. EBV infection is associated with c-met abnormal expression but not with c-myc protein in EBVaGC. c-met overexpression is not induced by LMP1. BARF1 and BHRF1 may play important roles in the tumorigenesis of EBVaGC through different pathways.
PMCID: PMC4087508  PMID: 16609989
Epstein-Barr virus; Helicobacter pylori; Gastric carcinoma; c-met; c-myc
14.  Epstein-Barr virus infection is inversely correlated with the expression of retinoblastoma protein in Reed-Sternberg cells in classic Hodgkin lymphoma 
Classic Hodgkin lymphoma (cHL) is characterized by few neoplastic Hodgkin/Reed-Sternberg (H/RS) cells in a background of intense inflammatory infiltrate. Epstein-Barr virus (EBV) has been shown to affect cell cycle and regulation of apoptosis. In total, 82 cases of cHL were studied. Five- micrometer sections were prepared and stained with haematoxylin and eosin and immunohistochemical streptavidin-biotin methods for EBV-LMP-1, pRb, ki-67 and cleaved caspase-3. In-situ hybridization for EBV encoded RNA was used to confirm the detection of EBV in H/RS cells. There were 45 nodular sclerosis, 28 mixed cellularity, 4 lymphocyte-rich, and 5 lymphocyte depletion subtypes in this series of cases. EBV and pRb were detected in 55% (46/82) and 64% (50/82) of the cases respectively. EBV was detected in 78% (25/32) of pRb-negative cases and 81% (29/36) of EBV-negative cases are pRb-positive. A statistically significant inverse relationship was observed between the presence of EBV and expression of pRb (P = 0.001). In conclusion, EBV infection is inversely correlated with pRb in H/RS cells in cHL.
PMCID: PMC4270594  PMID: 25550786
Hodgkin lymphoma; Epstein Barr virus; pRb
15.  Alterations in the human epidermal growth factor receptor 2-phosphatidylinositol 3-kinase-v-Akt pathway in gastric cancer 
AIM: To investigate human epidermal growth factor receptor 2 (HER2)-phosphatidylinositol 3-kinase (PI3K)-v-Akt murine thymoma viral oncogene homolog signaling pathway.
METHODS: We analyzed 231 formalin-fixed, paraffin-embedded gastric cancer tissue specimens from Japanese patients who had undergone surgical treatment. The patients’ age, sex, tumor location, depth of invasion, pathological type, lymph node metastasis, and pathological stage were determined by a review of the medical records. Expression of HER2 was analyzed by immunohistochemistry (IHC) using the HercepTestTM kit. Standard criteria for HER2 positivity (0, 1+, 2+, and 3+) were used. Tumors that scored 3+ were considered HER2-positive. Expression of phospho Akt (pAkt) was also analyzed by IHC. Tumors were considered pAkt-positive when the percentage of positive tumor cells was 10% or more. PI3K, catalytic, alpha polypeptide (PIK3CA) mutations in exons 1, 9 and 20 were analyzed by pyrosequencing. Epstein-Barr virus (EBV) infection was analyzed by in situ hybridization targeting EBV-encoded small RNA (EBER) with an EBER-RNA probe. Microsatellite instability (MSI) was analyzed by polymerase chain reaction using the mononucleotide markers BAT25 and BAT26.
RESULTS: HER2 expression levels of 0, 1+, 2+ and 3+ were found in 167 (72%), 32 (14%), 12 (5%) and 20 (8.7%) samples, respectively. HER2 overexpression (IHC 3+) significantly correlated with intestinal histological type (15/20 vs 98 /205, P = 0.05). PIK3CA mutations were present in 20 cases (8.7%) and significantly correlated with MSI (10/20 vs 9/211, P < 0.01). The mutation frequency was high (21%) in T4 cancers and very low (6%) in T2 cancers. Mutations in exons 1, 9 and 20 were detected in 5 (2%), 9 (4%) and 7 (3%) cases, respectively. Two new types of PIK3CA mutation, R88Q and R108H, were found in exon1. All PIK3CA mutations were heterozygous missense single-base substitutions, the most common being H1047R (6/20, 30%) in exon20. Eighteen cancers (8%) were EBV-positive and this positivity significantly correlated with a diffuse histological type (13/18 vs 93/198, P = 0.04). There were 7 cases of lymphoepithelioma-like carcinomas (LELC) and 6 of those cases were EBV-positive (percent/EBV: 6/18, 33%; percent/all LELC: 6/7, 86%). pAkt expression was positive in 119 (53%) cases but showed no correlation with clinicopathological characteristics. pAkt expression was significantly correlated with HER2 overexpression (16/20 vs 103/211, P < 0.01) but not with PIK3CA mutations (12/20 vs 107/211, P = 0.37) or EBV infection (8/18 vs 103/211, P = 0.69). The frequency of pAkt expression was higher in cancers with exon20 mutations (100%) than in those with exon1 (40%) or exon9 (56%) mutations. One case showed both HER2 overexpression and EBV infection and 3 cases showed both PIK3CA mutations and EBV infection. However, no cases showed both PIK3CA mutations and HER2 overexpression. One EBV-positive cancer with PIK3CA mutation (H1047R) was MSI-positive. Three of these 4 cases were positive for pAkt expression. In survival analysis, pAkt expression significantly correlated with a poor prognosis (hazard ratio 1.75; 95%CI: 1.12-2.80, P = 0.02).
CONCLUSION: HER2 expression, PIK3CA mutations and EBV infection in gastric cancer were characterized. pAkt expression significantly correlates with HER2 expression and with a poor prognosis.
PMCID: PMC3516204  PMID: 23236232
Human epidermal growth factor receptor 2; Phosphatidylinositol 3-kinase; Catalytic; Alpha polypeptide; Epstein-Barr virus; Akt; Gastric cancer
16.  Epstein-Barr virus-associated lymphoproliferative disorder developed following autologous peripheral blood stem cell transplantation for relapsing Hodgkin’s lymphoma 
Oncology Letters  2012;3(6):1203-1206.
Post-transplant lymphoproliferative disorders (PTLDs) are lymphoid or plasmacytic proliferations that develop as a consequence of immunosuppression in a recipient of a solid organ, bone marrow or stem cell allograft. The development of PTLDs is usually associated with Epstein-Barr virus (EBV) and the disorder is also termed EBV-associated lymphoproliferative disorder (LPD). The development of PTLD is a rare complication in autologous bone marrow/peripheral blood stem cell transplantation. In the present study, we report a case of EBV-associated LPD which developed following autologous peripheral blood stem cell transplantation for relapsing Hodgkin’s lymphoma. A 51-year-old male presented with swelling of the left cervical lymph nodes. A biopsy revealed nodular sclerosis classical Hodgkin’s lymphoma. Following four courses of ABVd (adriamycin, bleomycin, vinblastine, dacarbazine) therapy, the Hodgkin’s lymphoma relapsed. CHASE (cyclophosphamide, etoposide, cytarabine, dexamethasone) therapy and autologous peripheral blood stem cell transplantation were performed. In the 128 days following the transplantation, lymph node swelling was noted and a biopsy specimen demonstrated EBV-associated LPD. The serum copy number of EBV-DNA was 2.7×103 copies/ml. The occurrence of EBV-associated LPD may be on the rise due to the increased number of patients undergoing immunosuppression therapy. The measurement of the serum EBV-DNA copy number and the detection of EBV-infected atypical lymphocytes using in situ hybridization are significant in establishing an early accurate diagnosis and initiating the correct treatment for EBV-associated LPD in patients with immunosuppression.
PMCID: PMC3392596  PMID: 22783418
Epstein-Barr virus; lymphoproliferative disorder; Hodgkin’s lymphoma; autologous peripheral blood stem cell transplantation
17.  Analysis of Epstein-Barr virus reservoirs in paired blood and breast cancer primary biopsy specimens by real time PCR 
Breast Cancer Research  2006;8(6):R70.
Epstein-Barr virus (EBV) is present in over 90% of the world's population. This infection is considered benign, even though in limited cases EBV is associated with infectious and neoplastic conditions. Over the past decade, the EBV association with breast cancer has been constantly debated. Adding to this clinical and biological uncertainty, different techniques gave contradictory results for the presence of EBV in breast carcinoma specimens. In this study, minor groove binding (MGB)-TaqMan real time PCR was used to detect the presence of EBV DNA in both peripheral blood and tumor samples of selected patients.
Peripheral blood and breast carcinoma specimens from 24 patients were collected. DNA was extracted and then amplified by MGB-TaqMan real time PCR.
Of 24 breast tumor specimens, 11 (46%) were positive for EBV DNA. Of these 11 breast tumor specimens, 7 (64%) were also positive for EBV DNA in the peripheral blood, while 4 (36%) were positive for EBV DNA in the tumor, but negative in the blood.
EBV was found at extremely low levels, with a mean of 0.00004 EBV genomes per cell (range 0.00014 to 0.00001 EBV genomes per cell). Furthermore, our finding of the presence of EBV in the tumor specimens coupled to the absence of detection of EBV genomic DNA in the peripheral blood is consistent with the epithelial nature of the virus. Because of the low levels of viral DNA in tumor tissue, further studies are needed to assess the biological input of EBV in breast cancer.
PMCID: PMC1797024  PMID: 17163997
18.  Epstein-Barr Virus (EBV) detection and typing by PCR: a contribution to diagnostic screening of EBV-positive Burkitt's lymphoma 
Diagnostic Pathology  2006;1:17.
Epstein-Barr virus (EBV) is associated to the etio-pathogenesis of an increasing number of tumors. Detection of EBV in pathology samples is relevant since its high prevalence in some cancers makes the virus a promising target of specific therapies. RNA in situ hybridization (RISH) is the standard diagnostic procedure, while polymerase chain reaction (PCR)-based methods are used for strain (EBV type-1 or 2) distinction. We performed a systematic comparison between RISH and PCR for EBV detection, in a group of childhood B-cell Non-Hodgkin lymphomas (NHL), aiming to validate PCR as a first, rapid method for the diagnosis of EBV-associated B-cell NHL.
EBV infection was investigated in formalin fixed paraffin-embedded tumor samples of 41 children with B-cell NHL, including 35 Burkitt's lymphoma (BL), from Rio de Janeiro, Brazil, by in situ hybridization of EBV-encoded small RNA (EBER-RISH) and PCR assays based on EBNA2 amplification.
EBV genomes were detected in 68% of all NHL. Type 1 and 2 accounted for 80% and 20% of EBV infection, respectively. PCR and RISH were highly concordant (95%), as well as single- and nested-PCR results, allowing the use of a single PCR round for diagnostic purposes. PCR assays showed a sensitivity and specificity of 96% and 100%, respectively, with a detection level of 1 EBV genome in 5,000–10,000 EBV-negative cells, excluding the possibility of detecting low-number EBV-bearing memory cells.
We describe adequate PCR conditions with similar sensitivity and reliability to RISH, to be used for EBV diagnostic screening in high grade B-NHL, in "at risk" geographic regions.
PMCID: PMC1559641  PMID: 16893464
19.  HIV Patients Developing Primary CNS Lymphoma Lack EBV-Specific CD4+ T Cell Function Irrespective of Absolute CD4+ T Cell Counts 
PLoS Medicine  2007;4(3):e96.
In chronic HIV infection, antiretroviral therapy–induced normalization of CD4+ T cell counts (immune reconstitution [IR]) is associated with a decreased incidence of opportunistic diseases. However, some individuals remain at risk for opportunistic diseases despite prolonged normalization of CD4+ T cell counts. Deficient Epstein-Barr virus (EBV)-specific CD4+ T cell function may explain the occurrence of EBV-associated opportunistic malignancy—such as primary central nervous system (PCNS) lymphoma—despite recovery of absolute CD4+ T cell counts.
Methods and Findings
Absolute CD4+ T cell counts and EBV-specific CD4+ T cell-dependent interferon-γ production were assessed in six HIV-positive individuals prior to development of PCNS lymphoma (“cases”), and these values were compared with those in 16 HIV-infected matched participants with no sign of EBV-associated pathology (“matched controls”) and 11 nonmatched HIV-negative blood donors. Half of the PCNS lymphoma patients fulfilled IR criteria (defined here as CD4+ T cell counts ≥500/μl blood). EBV-specific CD4+ T cells were assessed 0.5–4.7 y prior to diagnosis of lymphoma. In 0/6 cases versus 13/16 matched controls an EBV-specific CD4+ T cell response was detected (p = 0.007; confidence interval for odds ratio [0–0.40]). PCNS lymphoma patients also differed with regards to this response significantly from HIV-negative blood donors (p < 0.001, confidence interval for odds ratio [0–0.14]), but there was no evidence for a difference between HIV-negative participants and the HIV-positive matched controls (p = 0.47).
Irrespective of absolute CD4+ T cell counts, HIV-positive patients who subsequently developed PCNS lymphoma lacked EBV-specific CD4+ T cell function. Larger, ideally prospective studies are needed to confirm these preliminary data, and clarify the impact of pathogen-specific versus surrogate marker-based assessment of IR on clinical outcome.
In a case-control study from the Swiss HIV cohort, Hess and colleagues report that T-helper responses against Epstein-Barr virus are specifically absent in patients developing CNS lymphoma.
Editors' Summary
AIDS causes disease by inactivating the body's immune responses. Most severely affected are the white blood cells known as T lymphocytes, particularly the CD4+ T cells that recognize infection and enable other cells of the immune system to respond. Advanced HIV infection, marked by very low numbers of CD4+ cells, is associated with a variety of infections and tumors that are rarely seen in people with intact immune systems. People with advanced HIV who receive highly active antiretroviral treatment (HAART) tend to have increases in their CD4+ cell counts and lose their susceptibility to these so-called opportunistic infections and cancers. For several common opportunistic infections, it is considered safe to discontinue preventive antibiotics after a patient's total CD4+ cell count has returned to normal levels on HAART. Some treated individuals, however, will develop these conditions even after their CD4+ cell counts have returned to normal levels. The reason this happens is unclear.
Why Was This Study Done?
For several years, scientists have speculated that susceptibility to a given opportunistic infection might be due not simply to low total CD4+ cells, but to loss of the specific CD4+ cells that recognize the infection in question. If this theory is correct, then those individuals who develop an opportunistic condition after their total CD4+ cell counts return to normal might be missing the specific cells that respond to the microbe causing the condition. The researchers wanted to test this theory in HIV patients with a brain tumor called primary central nervous system lymphoma (PCNS lymphoma). The Epstein-Barr virus (EBV), which causes mononucleosis in the general population, has been shown to be a cause of PCNS lymphoma in people with AIDS.
What Did the Researchers Do and Find?
The researchers studied patients who developed PCNS lymphoma while enrolled in the Swiss HIV Cohort, an ongoing study that has enrolled more than 14,000 people. A large cohort was needed to address this question because PCNS lymphoma is uncommon, and indeed only six patients with a confirmed diagnosis were identified. Because they had been followed as part of the cohort study, these patients had given blood samples that could be tested in retrospect. Three of these patients had low CD4+ cell counts prior to lymphoma diagnosis and three had normal CD4+ cell counts, but CD4 responses specifically against EBV were absent or very low in all six patients before they were diagnosed with PCNS lymphoma. The researchers also studied a comparison group of cohort participants with comparable CD4+ cell counts but no PCNS lymphoma, and found that 13/16 of those participants did have CD4 responses to EBV.
What Do These Findings Mean?
These results support the idea that the action of EBV-specific CD4+ cells, rather than a given level of total CD4+ cells, is needed to prevent PCNS lymphoma. Because only a small number of cases were identified, this must be considered a preliminary result. Given the rarity of PCNS lymphoma, however, especially in people receiving HAART, it seems unlikely that a larger cohort will be available in the near future to provide a more definitive conclusion. Based on this result, it may be useful to perform similar studies of other opportunistic infections. If a “gap” in the CD4+ cell response can be shown to increase the risk of a specific condition, it may become appropriate to test specific CD4 responses before deciding to discontinue preventive treatment as CD4+ cell counts increase on HAART.
Additional Information.
Please access these Web sites via the online version of this summary at
Read the accompanying Perspective by Mark Jacobson, MD
The Swiss Cohort Study Web site contains information on related research projects
The UCSF Center for HIV Information's HIV InSite includes resources on HIV immunology and opportunistic infections
PMCID: PMC1831733  PMID: 17388662
20.  Epstein-Barr virus in oral shedding of children with multiple sclerosis 
Neurology  2013;81(16):1392-1399.
To investigate Epstein-Barr virus (EBV) oral shedding frequency and EBV genetic diversity in pediatric patients with multiple sclerosis (MS).
This was a prospective case-control study. We used PCR-based assays to detect viral DNA in the monthly mouth swabs of 22 pediatric patients with MS and 77 age- and sex-matched healthy controls. EBV-positive samples were further analyzed for sequence variation in the EBV BCRF1 (ebvIL-10) gene using direct DNA sequencing methods, and in the EBV LMP1 gene by mass spectrometry.
Nineteen of the 22 (86.4%) children with MS were seropositive for remote EBV infection compared to 35 out of 77 (45.5%) healthy controls (p = 0.008). Baseline analysis of mouth swabs revealed a higher proportion of EBV-positive samples from EBV-seropositive patients with MS compared to EBV-seropositive healthy controls (52.6% vs 20%, p = 0.007). Longitudinal analysis of monthly swabs revealed average EBV detection rates of 50.6% in patients with MS and 20.4% in controls (p = 0.01). The oral shedding frequencies of Herpesviruses herpes simplex virus–1, cytomegalovirus, human herpesvirus (HHV)-6, and HHV-7 did not differ between groups. Changes in the predominant EBV genetic variants were detected more frequently in patients with MS; however, no specific EBV genetic variant was preferentially associated with MS.
Children with MS demonstrate abnormally increased rates of EBV viral reactivation and a broader range of genetic variants, suggesting a selective impairment in their immunologic control of EBV.
PMCID: PMC3806908  PMID: 24014504
21.  Epstein–Barr virus expression in Hodgkin’s disease in relation to patient characteristics, serum factors and blood lymphocyte function 
British Journal of Cancer  1999;81(7):1182-1187.
Epstein–Barr virus (EBV) expression was investigated by immunohistochemistry (latent membrane protein 1 [LMP-1]) and in situ hybridization (EBV encoded RNA [EBER]) in biopsies from 95 patients with untreated Hodgkin’s disease (HD). Tumour EBV status was related to EBV antibody titres, spontaneous and concanavallin A induced blood lymphocyte DNA synthesis, serum levels of soluble (s) CD4, sCD8, sCD25, sCD30, sCD54, β2-microglobulin, thymidine-kinase, routine chemistry, patient characteristics, complete remission and survival. The median follow-up time was 145 months (range 60–257). Tumour EBV-positive (n = 30; 33%) and negative (n = 62; 67%) patients did not differ with regard to sex, age, stage, presence of bulky disease or B-symptoms, remission rate or survival. The proportion of EBV+ cases was significantly higher among patients with mixed cellularity histopathology (58%) as compared to the nodular sclerosis subtype (18%; P < 0.001). The total white blood cell (WBC) counts were significantly lower in EBV+ patients (P < 0.01), who also had significantly higher levels of sCD54 (P < 0.02) and a tendency towards lower levels of sCD30 (P = 0.056). Patients in the tumour EBV+ group had significantly higher IgG antibody titres to restricted early antigen (EA-R) (P < 0.02). Hence, clinical features and outcome were not related to tumour EBV status. However, HD patients with EBV+ tumours had elevated sCD54 levels, higher antibody titres to EA-R and decreased total WBC counts. A potential causal relationship between EBV tumour status and these findings needs to be further explored. © 1999 Cancer Research Campaign
PMCID: PMC2374328  PMID: 10584880
Epstein–Barr virus; Hodgkin’s disease; lymphocyte function; prognosis; sCD30; s-ICAM-1
22.  Epstein-Barr Virus Infection is Common in Inflamed Gastrointestinal Mucosa 
Digestive diseases and sciences  2012;57(7):1887-1898.
Background and Aims
Epstein-Barr virus (EBV) is present in the malignant epithelial cells of 10% of all gastric adenocarcinomas, however localization of the virus in normal gastrointestinal mucosa is largely unexplored. In the current study, we measured EBV DNA and localized viral gene products in gastritis specimens (n=89), normal gastric and colonic mucosa (n=14), Crohn’s disease (n=9), and ulcerative colitis (n=11) tissues.
A battery of sensitive and specific quantitative polymerase chain reactions targeted six disparate regions of the EBV genome: BamH1W, EBNA1, LMP1, LMP2, BZLF1, and EBER1. EBV infection was localized by EBV-encoded RNA (EBER) in situ hybridization and by immunohistochemical stains for viral latent proteins LMP1 and LMP2 and for viral lytic proteins BMRF1 and BZLF1. B lymphocytes were identified using CD20 immunostains.
EBV DNA was essentially undetectable in normal gastric mucosa but was present in 46% of gastritis lesions, 44% of normal colonic mucosa, 55% of Crohn’s disease, and 64% of ulcerative colitis samples. Levels of EBV DNA exceeded what would be expected based on the numbers of B lymphocytes in inflamed tissues, suggesting that EBV is preferentially localized to inflammatory gastrointestinal lesions. Histochemical staining revealed EBER expression in lymphoid cells of some PCR-positive lesions. The viral lytic viral proteins, BMRF1 and BZLF1, were expressed in lymphoid cells of two ulcerative colitis tissues, both of which had relatively high viral loads by quantitative PCR.
EBV-infected lymphocytes are frequently present in inflamed gastric and colonic mucosa. Active viral replication in some lesions raises the possibility of virus-related perpetuation of gastrointestinal inflammation.
PMCID: PMC3535492  PMID: 22410851
Epstein-Barr virus; Gastritis; Colitis; Inflammation; Gene expression; Viral load
23.  Epstein-Barr Virus and p16INK4A Methylation in Squamous Cell Carcinoma and Precancerous Lesions of the Cervix Uteri 
Journal of Korean Medical Science  2005;20(4):636-642.
Methylation of p16 is an important mechanism in cervical carcinogenesis. However, the relationship between cervical squamous cell carcinoma (SCC) and Epstein-Barr virus (EBV) remains controversial. Here, we explored whether EBV infection and/or p16 gene inactivation would play any role in cervical carcinogenesis. Eighty-two specimens included 41 invasive SCCs, 30 cervical intraepithelial neoplasm (CIN; CIN 1, 11 cases, CIN II, 3 cases, CIN III 16 cases) and 11 nonneoplastic cervices. EBV was detected by polymerase chain reaction (PCR) for EBNA-1 and in situ hybridization for EBER-1. The p16 methylation-status and the expression of p16 protein were studied by methylation-specific PCR and immunohistochemistry, respectively. The materials were divided into four groups: 1) nonneoplastic cervices, 2) CIN I, 3) CIN II-III and 4) invasive SCCs. p16 methylation and p16 immunoexpressions increased in CIN and invasive SCCs than nonneoplastic tissue. p16-methylation and p16-immunoreactivities were higher in the EBV-positive group (p=0.009, p<0.001) than in the EBV-negative group. EBV was detected more frequently in CIN and SCCs than nonneoplastic cervices. In conclusion, a correlation between p16 methylation, p16 immunoreactivity and the detection of EBV strongly suggested that the cooperation of EBV and p16 gene may play a synergic effect on cell cycle deregulation.
PMCID: PMC2782161  PMID: 16100457
Protein p16; Methylation; Herpesvirus 4, Human; Epstein-Barr Virus Infections; Cervix Neoplasms; Carcinogenesis
24.  Epstein-Barr Virus and Human Papillomavirus Infections and Genotype Distribution in Head and Neck Cancers 
PLoS ONE  2014;9(11):e113702.
To investigate the prevalence, genotypes, and prognostic values of Epstein-Barr virus (EBV) and human papillomavirus (HPV) infections in Japanese patients with different types of head and neck cancer (HNC).
Methods and Materials
HPV and EBV DNA, EBV genotypes and LMP-1 variants, and HPV mRNA expression were detected by PCR from fresh-frozen HNC samples. HPV genotypes were determined by direct sequencing, and EBV encoded RNA (EBER) was examined by in situ hybridization.
Of the 209 HNC patients, 63 (30.1%) had HPV infection, and HPV-16 was the most common subtype (86.9%). HPV E6/E7 mRNA expression was found in 23 of 60 (38.3%) HPV DNA-positive cases detected. The site of highest prevalence of HPV was the oropharynx (45.9%). Among 146 (69.9%) HNCs in which EBV DNA was identified, 107 (73.3%) and 27 (18.5%) contained types A and B, respectively, and 124 (84.9%) showed the existence of del-LMP-1. However, only 13 (6.2%) HNCs were positive for EBER, 12 (92.3%) of which derived from the nasopharynx. Co-infection of HPV and EBER was found in only 1.0% of HNCs and 10.0% of NPCs. Kaplan-Meier survival analysis showed significantly better disease-specific and overall survival in the HPV DNA+/mRNA+ oropharyngeal squamous cell carcinoma (OPC) patients than in the other OPC patients (P = 0.027 and 0.017, respectively). Multivariate analysis showed that stage T1–3 (P = 0.002) and HPV mRNA-positive status (P = 0.061) independently predicted better disease-specific survival. No significant difference in disease-specific survival was found between the EBER-positive and -negative NPC patients (P = 0.155).
Our findings indicate that co-infection with HPV and EBV is rare in HNC. Oropharyngeal SCC with active HPV infection was related to a highly favorable outcome, while EBV status was not prognostic in the NPC cohort.
PMCID: PMC4236156  PMID: 25405488
25.  Expression of Epstein-Barr virus genes in EBV-associated gastric carcinomas 
AIM: To understand the expression of latent and lytic genes of Epstein-Barr virus (EBV) in EBV-associated gastric carcinoma (EBVaGC) and to explore the relationship between EBV-encoded genes and development of EBVaGC at molecular level.
METHODS: One hundred and seventy-two gastric carcinoma tissues and 172 corresponding para-carcinoma tissues were tested for EBV genome by polymerase chain reaction (PCR)-Southern blotting. EBV-encoded small RNA (EBER) 1 of the PCR positive specimens was detected by in situ hybridization (ISH). Gastric carcinomas with positive EBER1 signals were classified as EBVaGCs. RT-PCR and Southern hybridization were applied to the detection of expression of nuclear antigen (EBNA) promoters (Qp, Wp and Cp), EBNA 1 and EBNA 2, latent membrane proteins (LMP) 1, 2A and 2B and lytic genes (immediate early genes BZLF1 and BRLF1, early genes BARF1 and BHRF1, late genes BcLF1 and BLLF1) in EBVaGCs.
RESULTS: Eleven EBV positive samples existed in gastric carcinoma tissues (6.39%). No EBV positive sample was found in corresponding para-carcinoma tissues. The difference between EBV positivity in carcinoma tissues and corresponding para-carcinoma tissues was significant (χ2 = 9.0909, P = 0.0026). Transcripts of Qp and EBNA1 were detected in all the 11 EBVaGCs, while both Wp and Cp were silent. EBNA2, LMP1 and LMP2B mRNA were absent in all the cases, while LMP2A mRNA was detected in 4 of the 11 cases. Of the 11 EBVaGCs, 7 exhibited BcLF1 transcripts and 2 exhibited BHRF1 transcripts. The transcripts of BZLF1 and BARF1 were detected in 5 cases, respectively. No BLLF1 and BRLF mRNA were detected.
CONCLUSION: The latent pattern of EBV in gastric carcinoma corresponds to the latency I/II. Some lytic infection genes are expressed in EBVaGCs tissues. BARF1 and BHRF1 genes may play an important role in tumorigenesis of gastric carcinoma.
PMCID: PMC4250728  PMID: 15655811
Gastric carcinomas; Epstein-Barr virus; Gene expression

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